Alpinumisoflavone induces apoptosis in esophageal squamous cell carcinoma by modulating miR-370/PIM1 signaling

Abstract

Esophageal squamous cell carcinoma (ESCC) is the most prevalent type of esophageal cancer and accumulating evidence has confirmed the role of miRNAs in ESCC. One such miRNA, miR-370, was found to be aberrantly downregulated in various human malignancies. This study showed that the expression of miR-370 was significantly lower in ESCC tissues and cell lines, and miR-370 functioned as a tumor suppressor in ESCC. Moreover, this is the first report that showed miR-370 suppresses cell proliferation and tumor growth by directly targeting Pim family kinases 1 (PIM1). Furthermore, alpinumisoflavone, a naturally occurring flavonoid, could inhibit tumor growth of ESCC by targeting miR-370/PIM1 signaling.

Keywords: ESCC; PIM1; alpinumisoflavone; miR-370.

Figure 1

MiR-370 level in ESCC tissues. A. MiR-370 expression is lower in ESCC tissue as compared to adjacent normal tissue. B. MiR-370 expression is associated with TNM stage. C. MiR-370 expression is associated with pathological stage. **P<0.01.

Figure 2

MiR-370 functions as a tumor suppressor in ESCC cells. A. MiR-370 is significantly downregulated in ESCC cells. B. The level of miR-370 is manipulated using miR-370 mimic and miR-370 inhibitor. C. MiR-370 mimic and inhibitor affected the viability of ESCC cells. D. Mi-370 mimic suppresses cell proliferation, while miR-370 inhibitor promotes cell growth. E. MiR-370 mimic induces apoptosis, while miR-370 inhibitor suppresses apoptosis in ESCC cells. F. MiR-370 mimic activates caspase-3, while miR-370 inhibitor suppresses caspase-3 activation in ESCC cells. **P<0.01.

Figure 3

MiR-370 directly targets the PIM1 gene. A. Schematic illustration of the hypothesized duplexes formed from interactions between the PIM1 3’-UTR binding sites and miR-370. B. Inverse correlation between miR-370 and PIM1 mRNA level in ESCC tissues. C. Inverse correlation between miR-370 and PIM1 protein level in ESCC tissues. D. Quantitative analysis of PIM1 mRNA levels after transfection with miR-370 mimic and miR-370 inhibitor. E. Quantitative analysis of PIM1 protein levels after transfection with miR-370 mimic and miR-370 inhibitor. F. Luciferase activity is increased by miR-370 inhibitor and suppressed by miR-370 mimic. **P<0.01.

Figure 4

PIM1 shRNA impairs the pro-growth effect of miR-370 inhibitor in ESCC cells. A. PIM1 expression is suppressed by targeted shRNA. B. Knockdown of PIM1 in cells transfected with miR-370 inhibitor results in loss of cell viability. C. Knockdown of PIM1 in cells transfected with miR-370 inhibitor leads to growth inhibition. D. Knockdown of PIM1 in cells transfected with miR-370 inhibitor results in increased apoptosis. E. Knockdown of PIM1 in cells transfected with miR-370 inhibitor results in caspase-3 activation. **P<0.01.

Figure 5

PIM1 overexpression compromises the anti-tumor effect of miR-370 mimic in ESCC cells. A. PIM1 expression is increased by PIM1 vector. B. Overexpression of PIM1 in cells transfected with miR-370 mimic leads to impaired anti-proliferative effect. C. Overexpression of PIM1 in cells transfected with miR-370 mimic leads to increased cell growth. D. Overexpression of PIM1 in cells transfected with miR-370 mimic leads to apoptosis inhibition. E. Overexpression of PIM1 in cells transfected with miR-370 mimic correlates with decreased caspase-3 activity. **P<0.01.

Figure 6

AIF exerts anti-tumor effect in ESCC cells. A. AIF dose-dependently inhibits the proliferation of ESCC cells. B. AIF suppresses cell growth in a dose-dependent manner. C. AIF induces apoptosis in ESCC cells. D. AIF activates caspase-3 in a dose-dependent manner. **P<0.01.

Figure 7

AIF upregulates miR-370 and represses the expression of PIM1. A. AIF dose-dependently increases the expression level of miR-370. B. AIF dose-dependently suppresses the mRNA level of PIM1. C. AIF dose-dependently represses the protein level of PIM1. D. MiR-370 inhibitor or PIM1 overexpression attenuates the anti-proliferative effect of AIF. E. MiR-370 inhibitor or PIM1 overexpression significantly abrogates the anti-growth effect of AIF. F. MiR-370 inhibitor or PIM1 overexpression compromises the apoptosis-inducing effect of AIF. G. MiR-370 inhibitor or PIM1 overexpression impairs the suppressing effect of AIF on invasion. **P<0.01.

Figure 8

AIF exerts anti-tumor effect in xenograft model of ESCC. A. AIF treatment does not change the body weight of the animal model. B. AIF suppresses tumor growth in a dose-dependent manner. C. AIF induces apoptosis in vivo. D. AIF activates caspase-3 in vivo. E. AIF elevates the expression of miR-370 in tumor tissue. F. AIF suppresses the expression of PIM1 in tumor tissue. *P<0.05, **P<0.01.